This invention relates to a method and a dry analytical element for the determination of bicarbonate ion existing in a liquid sample. The method and dry analytical element are particularly effective for the determination of bicarbonate ion in a sample in clinical assay, such as blood or urine, which requires rapidity and high accuracy.
A conventional method for the determination of bicarbonate ion is of measuring the partial pressure of carboxylic acid in a liquid and hydrogen ion concentration (pH) by using electrodes. The concentration of bicarbonate ion can be determined by calculating from the above values. However, the method is disadvantageous in the simultaneous measurement of the partial pressure of carboxylic acid and pH of the liquid.
Another conventional method is of utilizing the conversion of bicarbonate ion into carbon dioxide in acidic conditions, and measuring the volume of the evolved carbon dioxide. In general, a large scale equipment is necessary for measuring the volume of gas accurately, and accordingly, this method is disadvantageous to the measurement of a large number of samples.
In order to solve the above disadvantages, some enzyme methods were developed.
The enzyme method disclosed in Japanese Patent KOKAI 4-210599 utilizes the following reactions, ##STR1## and bicarbonate ion is determined by measuring the decrease of absorption at 340 nm of NAD(P)H. In the above formulas, PEPC is phosphoenolpyruvate carboxylase, PEP is phosphoenolpyruvic acid, MDH is malate dehydrogenase, NADH is nicotinamide adenine dinucleotide in reduced form, NADPH is nicotinamide adenine dinucleotide phosphate in reduced form, and NAD and NADP are their oxidized forms, respectively.
The method disclosed in Japanese Patent KOKAI 4-248997 uses phosphoenolpyruvate carboxykinase instead of PEPC.
However, the above methods have following problems. First, since the absorbance at 340 nm is measured, the analyzer to be used must be equipped with an ultraviolet light source and detecting system therefor. As a result, the analyzer has a large scale, and is expensive. Second, since the absorbance of NAD(P)H at 340 nm is great, it is difficult to incorporate a necessary amount of NAD(P)H for converting the whole oxaloacetic acid produced in the above reaction into malic acid from first, by considering reaction rate. As a result, the determination range is narrow. Third, the above great absorbane problem can be avoided by changing the measuring wave length to a range of lower absorbance, such as 380 nm, and incorporating a sufficient amount of NAD(P)H. However, the measurement becomes unstable there due to the spectrum being not flat but oblique.
Moreover, blood samples are sometimes hemolyzed. Once hemolysis occurs, carbonic anhydrase eluted out of blood cell converts bicarbonate ion in blood into carbon dioxide. This brings a minus error on measurement. A countermeasure therefor is to add acetazolamide which is an inhibitor of carbonic anhydrase (Japanese Patent KOKAI 4-210599). However, acetazolamide has a difficulty in handling due to its irritation against eye and skin, and high price is also a problem.